In this project, we focused our studies on the genetic basis both of poxvirus virulence and of host resistance to virus infection. The acquired knowledge should contribute toward development of safe, effective recombinant vaccinia virus vaccines for animal and human use. The genetic basis for ectromelia virus' narrow host range is poorly understood. Analyses of the tissue tropism genes CHO, K1L, and C7L revealed that the CHO gene is inactive and the K1L and C7L genes have only partial activity. Deletion of the K1L gene had no detectable effect on ectromelia virus pathogenesis in the mouse. These observations are consistent with the narrow host range of ectromelia. A newly identified EV ORF, predicted to encode a 28 kDa protein, was shown to interact with DNA~presumably through a zinc finger motif in the carboxyl terminal region of the protein. The protein was expressed at low levels in the infected cell, and preliminary evidence suggested a nuclear site of localization. A 28 kDa- mutant replicated normally in tested tissue culture cell lines, but at least 100-fold less efficiently in tissues from the infected mouse and spread more slowly from the primary site of infection to spleen and liver. The LD50 of the mutant virus was at least 1000-fold lower than the WT. This gene appears to be an important poxvirus virulence determinant in the mousepox model. Treatment with IFN-gamma allowed mouse macrophages to restrict the replication of ectromelia, vaccinia, and herpes simplex viruses while producing nitric oxide. Inhibitors of nitric oxide synthase blocked the macrophages' antiviral activity. Virus replication was diminished in epithelial cells transfected with a cDNA encoding inducible nitric oxide synthase or treated with organic compounds that generate nitric oxide. In mice, an inhibitor of nitric oxide synthase converted sublethal, resolving ectromelia virus infection into fulminant mousepox. Thus, induction of nitric oxide synthase was necessary and sufficient for a substantial antiviral effect of IFN-gamma in vitro and in vivo.